Lactoferrin compositions and methods of wound treatment

ABSTRACT

The present invention relates to lactoferrin compositions and methods of using the compositions to treat wounds. The compositions can be administered alone or in combination with other standard wound healing therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/410,981 filed on Sep. 16, 2002 which is incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to lactoferrin compositions and methods ofusing the compositions to treat wounds. The compositions can beadministered alone or in combination with other standard wound healingtherapies. Yet further, the compositions can also comprise a metalchelator.

BACKGROUND OF THE INVENTION

Relatively few biotechnology products have been developed for treatingwounds, such as partial-thickness burns. Most of the efforts have beendirected towards chronic wounds, which do require a proper level ofcellular growth factors for healing. The most conventional option ofchronic ulcer treatment involves sharp debridement to remove allnon-viable tissue, a non-weight-bearing regimen, moist saline dressingschanged twice daily at which times the skin around the ulcer arecleansed with mild soap and water. Current advanced treatment forchronic ulcers include growth factors, skin replacement therapy,enzymatic and mechanical debridement to clean ischemic tissue, moistwound dressings, non-antibiotic cleansers, antibiotics (Edmonds et al.,2000, Lipsky and Berendt 2000, Moulin et al., 1998, Mandracchia et al.,2001). However, current therapy for chronic wounds is not completelyeffective. In fact, Regranex™ gel or Becaplermin (recombinant-humanplatelet-derived growth factor-BB), the only biological product in themarket for chronic wounds (diabetic neuropathic ulcers) has shown only9-23% improvement over placebo and 4-22% improvement over good ulcercare alone (Mandracchia et al., 2001, Edmonds et al., 2000, Wieman1998). Thus, an effective treatment for wounds, chronic and/or acute, isneeded.

Lactoferrin is an immunomodulatory human protein expressed throughoutthe body and found in highest concentrations in milk and colostrums.Recombinant human lactoferrin (RhLF) is a recombinant glycoproteinproduced in Aspergillus niger (A. niger), a filamentous fungi. RhLF isstructurally identical in all material respects to native lactoferrinand has a wide array of functions related to host defense mechanisms.For example, lactoferrin has been reported to activate natural killer(NK) cells, induce colony stimulating activity, activatepolymorphonuclear neutrophils (PMN), regulate granulopoeisis, enhanceantibody-dependent cell cytotoxicity, stimulate lymphokine-activatedkiller (LAK) cell activity, and potentiate macrophage toxicity.

Recombinant human lactoferrin has previously been described as beingpurified after expression in a variety of prokaryotic and eukaryoticorganisms including aspergillus (U.S. Pat. No. 6,080,559), cattle (U.S.Pat. No. 5,919,913), rice, corn, Sacharomcyes (U.S. Pat. No. 6,228,614)and Pichia pastoris (U.S. Pat. Nos. 6,455,687, 6,277,817, 6,066,469).Also described are expression systems for the expression of full-lengthhuman lactoferrins (e.g., U.S. Pat. No. 6,100,054). In all cases, partof the teaching is expression of the full length cDNA and purificationof the intact protein whose N-terminal, after processing of the leaderpeptide, is the amino acid glycine. Nuijens et al. (U.S. Pat. No.6,333,311) separately describe variants of human lactoferrin but theirfocus is limited to deletion or substitution of arginine residues foundin the N-terminal domain of lactoferrin.

EDTA (ethylenediaminetetraacetic acid) is a synthetic compound which haswell known metal-binding characteristics. EDTA is most commonly used forchelation therapy, a treatment that involves repeated intravenousadministration of EDTA to pull toxins from the bloodstream. EDTAadministration is the medically accepted treatment for poisoning byheavy metals such as lead, mercury, arsenic and thallium and has beenapproved by the Food and Drug Administration (FDA) for this use.

EDTA has also been proposed as a treatment for heart disease. Proponentsof chelation therapy for heart disease claim that EDTA, in combinationwith oral vitamins and minerals, helps dissolve plaques and mineraldeposits associated with atherosclerosis. Although many Americans withheart disease have turned to EDTA chelation therapy to improve theircondition, the FDA has not approved this therapy as an alternativetreatment for heart disease. It is thought that EDTA chelation may helpstrengthen the immune system by sequestering impurities from thebloodstream.

The present invention is the first to develop a suitable delivery systemto deliver lactoferrin to treat acute and/or chronic ulcers or othertypes of wounds, such as burns. Yet further, the present invention isthe first to use lactoferrin in combination with a metal chelator totreat acute and/or chronic wounds.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a composition comprisinglactoferrin or N-terminal lactoferrin variant in which at least theN-terminal glycine residue is truncated or substituted. The compositioncan be used as a treatment for wound healing. The method of treatmentinvolves the administration of the lactoferrin composition, which may beapplied topically, orally or parenterally. The lactoferrin compositioncan also be administered in combination with standard wound healingtherapies.

An embodiment of the present invention comprises a lactoferrincomposition having an N-terminal lactoferrin variant. More specifically,the lactoferrin is recombinant lactoferrin variant. Such N-terminallactoferrin variants includes variants that at least lack the N-terminalglycine residue or contain a substitution at the N-terminal glycineresidue. The substitution can comprise substituting a natural orartificial amino acid residue for the N-terminal glycine residue. Forexample, the substitution can comprise substituting a positive aminoacid residue or a negative amino acid residue for the N-terminal glycineresidue or substituting a neutral amino acid residue other than glycinefor the N-terminal glycine residue. Other N-terminal lactoferrinvariants include lactoferrin lacking one or more N-terminal residues orhaving one or more substitutions in the N-terminal.

In specific embodiments, the N-terminal lactoferrin variant comprises atleast 1% of the lactoferrin composition, at least 5% of the lactoferrincomposition, at least 10% of the lactoferrin composition, at least 25%of the lactoferrin composition, at least 50% of the lactoferrincomposition or any range in between.

An embodiment of the present invention is a pharmaceutical compositioncomprising a therapeutically effective amount of a lactoferrincomposition and a pharmaceutically acceptable polymer having a viscosityin the range of about 1 to about 12,000,000 cP at room temperature,wherein the amount of lactoferrin is sufficient to provide animprovement in the wound. The lactoferrin is mammalian lactoferrin, suchas human or bovine. More specifically, the lactoferrin is recombinantlactoferrin. Still further, the lactoferrin composition comprises avariant thereof in that at least the N-terminal glycine is truncatedand/or substituted. The N-terminal lactoferrin variant comprises atleast 1% of the lactoferrin composition, at least 5% of the lactoferrincomposition, at least 10% of the lactoferrin composition, at least 25%of the lactoferrin composition, at least 50% of the lactoferrincomposition or any range in between.

In specific embodiments, the polymer is selected from the groupconsisting of vinyl polymer (i.e., polyacrylic acid, polymethacrylicacid, polyvinyl pyrrolidone and polyvinyl alcohol), polysaccharidepolymer (i.e., cellulose, cellulose derivatives, glycosaminoglycans,agar, pectin, alginic acid, dextran, starch, and chitosan),glycosaminoglycan polymer (i.e., hyaluronic acid, chondroitin,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratansulfate, heparin sulfate and heparin), protein polymer (i.e., collagen,gelatin and fibronectin), polyoxyethylene-polyoxypropylene polymer(i.e., polyoxyethylene-polyoxypropylene block copolymer) and acrylamidepolymer (i.e., polyacrylamide or polymethacrylamide). Preferably, thepolyoxyethylene-polyoxypropylene block copolymer is F88 or F127.

In further embodiments, the lactoferrin (lactoferrin or N-terminallactoferrin variant in which at least the N-terminal glycine residue istruncated or substituted) concentration of the pharmaceuticalcomposition is within the range of about 0.0001% (w/w) to about 30%(w/w). More particularly, the polymer concentration is about 0.5% (w/w)to about 3.0% (w/w) and the polymer has an average molecular weight ofabout 500 to about 13,000,000.

A preferred embodiment is a pharmaceutical composition comprising anamount of recombinant human lactoferrin (rhLF lactoferrin or anN-terminal lactoferrin variant thereof such that at least the N-terminalglycine residue is truncated or substituted) that is sufficient toprovide an improvement in a wound, and a polymer, wherein the polymer isselected from the group consisting of a vinyl polymer, polysaccharidepolymer, glycosaminoglycan polymer, protein polymer,polyoxyethylene-polyoxypropylene polymer, and acrylamide polymer,wherein the composition is an aqueous gel having a viscosity in therange of about 1 to about 12,000,000 cP at room temperature. The polymerconcentration is about 0.5% (w/w) to about 3.0% (w/w) and the polymerhas a molecular weight of about 500 to about 13,000,000.

Another preferred embodiment is a pharmaceutical composition comprisingan amount of a lactoferrin composition that is sufficient to provide animprovement in a wound and a pharmaceutically acceptable polymerselected from the group consisting of a vinyl polymer, polysaccharidepolymer, glycosaminoglycan polymer, protein polymer,polyoxyethylene-polyoxypropylene polymer, and acrylamide polymer havinga concentration in the range of about 0.5% (w/w) to about 3.0% (w/w) andhaving a molecular weight in the range of about 500 to about 13,000,000,wherein the composition is an aqueous gel having a viscosity in therange of about 1 to about 12,000,000 cP at room temperature.

Another embodiment of the present invention is a method of treating awound comprising the step of administering to a subject a lactoferrincomposition in an amount sufficient to provide an improvement in thewound. The lactoferrin composition is dispersed in a pharmaceuticallyacceptable carrier. In further embodiments, the lactoferrin compositionis administered in combination with other standard wound healingtherapies. The lactoferrin composition can be administered for at leastone week, six weeks, 12 weeks, 36 weeks, etc. or any range in between.

In further embodiments, a metal chelator dispersed in a pharmaceuticallyacceptable carrier may also be administered with the lactoferrincomposition. Preferred metal chelator include, but are not limited toethylenediaminetetraacetic acid (EDTA) or[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA). Morepreferably, the metal chelator is EDTA.

Exemplary wounds that can be treated include, but are not limited toskin wounds, bone wounds, internal wounds gastrointestinal wounds, oralwounds, ophthalmic wounds, surgical wounds, or any combination thereof.A skin wound can be a full-thickness skin wound or a partial-thicknessskin wound. In specific embodiments, the wound is further defined as achronic wound, for example, but not limited to diabetic ulcer, venousstasis ulcer, pressure ulcer, and infected wound. Still further, thewound is further defined as an acute wound. Exemplary acute woundsinclude, but are not limited to first degree burn, partial-thicknessburn, full-thickness burn, laceration, bullet wound, and infected wound.

In further embodiments, the lactoferrin is administered topically,orally or parenterally. Still further, an antacid may also beadministered in conjunction with said lactoferrin composition.

In specific embodiments, the amount of the lactoferrin composition(lactoferrin or N-terminal lactoferrin variant such that at least theN-terminal glycine residue is truncated or substituted) that isadministered is about 0.0001 μg to about 100 g per day. The amount ofthe EDTA that is administered is about 1 ng to about 1 g per day.

In further embodiments, the lactoferrin composition is a topical gel, asolution, capsule or a tablet having a lactoferrin concentration ofabout 0.0001% to about 30%. Topical gel is composed from a polymerselected from the group of consisting of a vinyl polymer, polysaccharidepolymer, glycosaminoglycan polymer, protein polymer,polyoxyethylene-polyoxypropylene polymer, and acrylamide polymer. Thepolymer concentration is about 0.5% (w/w) to about 3.0% (w/w) and thepolymer has a molecular weight of about 50,000 to about 13,000,000.

Another embodiment is a method of treating a wound comprising the stepof supplementing the local immune system in a subject by administeringtopically the amount of lactoferrin in the vicinity of the wound. Thelactoferrin results in the killing of bacteria infecting the wound.

A further embodiment is a method of enhancing the local immune system ina subject suffering from a wound comprising the step of administeringtopically to the subject a lactoferrin composition. Lactoferrin resultsin the killing of bacteria infecting the wound. Lactoferrin stimulatesthe production of a cytokine or a chemokine. Exemplary cytokines thatcan be stimulated by lactoferrin include, but are not limited tointerleukin-18 (IL-18), interleukin-12 (IL-12), granulocyte/macrophagecolony-stimulating factor (GM-CSF), and gamma interferon (IFN-γ).Exemplary chemokines include, but are not limited to macrophageinflammatory protein 3 alpha (MIP-3α), macrophage inflammatory protein 1alpha (MIP-1α), or macrophage inflammatory protein beta (MIP-1β).

The lactoferrin composition of the present invention can also result ininhibition of a cytokine or chemokine. The cytokine is selected from thegroup consisting of interleukin-2 (IL-2), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-10 (IL-10), and tumor necrosis factoralpha (TNF-α). Still further, the lactoferrin composition can alsoinhibit the production of matrix metalloproteinases (MMPs).

Still further, interleukin-18 or granulocyte/macrophagecolony-stimulating factor stimulates the production or activity ofimmune cells. The immune cells are selected from the group consisting ofT lymphocytes, natural killer cells, macrophages, dendritic cells, andpolymorphonuclear cells. More specifically, the polymorphonuclear cellsare neutrophils and the T lymphocytes are selected from the groupconsisting of CD4+, CD8+ and CD3+ T cells.

In a further embodiment, interleukin-18 or granulocyte/macrophagecolony-stimulating factor stimulates the production or activity of cellsinvolved in wound repair. The cells involved in wound repair areselected from the group consisting of keratinocytes, endothelial cells,fibroblasts, dendritic cells and myofibroblasts. The inhibition ofTNF-alpha further inhibits the migration and maturation of dendriticcells. The dendritic cells are Langerhans cells.

Another embodiment is a method of treating a wound comprising the stepof supplementing the systemic immune system in a subject by increasingthe amount lactoferrin in the systemic circulation by administering thelactoferrin composition via a parenteral route that is selected from thegroup consisting of intramuscular, intravenous, intraperitoneal,intraoccular, intraarticular, and a surgical field.

A further embodiment is a method of enhancing the systemic immune systemof a subject suffering from a wound comprising the step of parenterallyadministering to the subject a lactoferrin composition.

Another embodiment is a method of treating a wound comprising the stepof supplementing the mucosal immune system in a subject by increasingthe amount of lactoferrin in the gastrointestinal tract of the subjectby administering orally a lactoferrin composition.

Still further, another embodiment is a method of enhancing the mucosalimmune system in a subject suffering from a wound comprising orallyadministering to the subject a lactoferrin composition.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 shows the incidence of 75% wound closure with and withoutadministration of recombinant human lactoferrin carbopol gels in healthyand diabetic mice.

FIG. 2A and FIG. 2B show the time course of wound healing with andwithout topical administration of recombinant human lactoferrin solutionin healthy mice. CGS-21680 (FIG. 2A) or Regranex™ (FIG. 2B) was used asa positive control. In FIG. 2A, * refers to significant compared tobuffer (p<0.05), ** refers to very significant compared to buffer(p<0.01) and # refers to significant compared to CGS-21680 (p<0.05). InFIG. 2B, * refers to significant compared to buffer (p<0.05), ′ refersto significant compared to rhPDGF [Regranex™] (p<0.05), ** verysignificant compared to buffer (p<0.01), and ″ refers to verysignificant compared to rhPDGF [Regranex™] (p<0.01).

FIG. 3 shows the effect of topical rhLF on the incidence of 75% woundclosure in healthy mice. Regranex ™ was used as a positive control.

FIG. 4A, FIG. 4B and FIG. 4C show the effect of oral rhLF on theincidence of 75% wound closure in healthy (FIG. 4A) and diabetic (FIG.4B) mice, and on the incidence of 100% wound closure in diabetic mice(FIG. 4C).

FIG. 5A and FIG. 5B show the incidence of 75% wound closure in aninfected wound healed with and without topical (FIG. 5A) or oral (FIG.5B) administration of recombinant human lactoferrin solution. Regranex™was used as a topical positive control (FIG. 5A).

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

A. Definitions

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.”

The term “acute wound” as used herein refers to a wound that heals in ashort amount of time. Examples of acute wounds include, but are notlimited to partial-thickness burn, laceration, bullet wound or infectedwound.

The term “chronic wound” as used herein refers to wounds that take along time to heal or that do not heal without external intervention. Yetfurther, as used herein, a “chronic wound”, also referred to as “chroniculcer” can be broadly classified into three major types: diabeticulcers, venous stasis ulcers, decubitus or pressure ulcers. Stillfurther, a chronic wound can also include infected wounds that take along time to heal.

The term “cytokine” as used herein refers to proteins that are made bycells that affect the behavior of other cells, for example stimulate orinhibit cell proliferation. For example, cytokines that are made bylymphocytes are often called lymphokines or interleukins. One of skillin the art realizes that the term cytokine is a generic term used in theliterature to refer to proteins that are made by cells that can effectthe behavior of other cells.

The term “chemokine” as used herein refers to small cytokines that areinvolved in the migration and activation of cells, for examplephagocytic cells and lymphocytes. One of skill in the art realizes thatchemokines play a central role in inflammatory and immune responseprocesses.

The term “lactoferrin” or “LF” as used herein refers to native orrecombinant lactoferrin. Native lactoferrin can be obtained bypurification from mammalian milk or colostrum or from other naturalsources. Recombinant lactoferrin (rLF) can be made by recombinantexpression or direct production in genetically altered animals, plants,fungi, bacteria, or other prokaryotic or eukaryotic species, or throughchemical synthesis.

The term “lactoferrin composition” as used herein refers to acomposition having lactoferrin or a part thereof, wherein at least theN-terminal glycine residue is truncated or substituted.

The term “metal chelator” as used herein refers to a compound whichbinds metal. Metal chelators that can be used in the present inventioninclude the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof.

The term “N-terminal lactoferrin variant” as used herein refers tolactoferrin wherein at least the N-terminal glycine has been truncatedand/or substituted. N-terminal lactoferrin variants also include, butare not limited to deletion and/or substitution of one or moreN-terminal amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 N-terminal amino acid residues, etc. Thus,N-terminal lactoferrin variants comprise at least deletions ortruncations and/or substitutions of 1 to 16 N-terminal amino acidresidues. The deletion and/or substitution of at least the N-terminalglycine of lactoferrin mediates the same biological effects asfull-length lactoferrin and/or may enhance lactoferrin's biologicalactivity, for example by stimulating the production of various cytokines(i.e., IL-18, MIP-3α, GM-CSF or IFN-γ), by inhibiting various cytokines,(i.e., IL-2, IL-4, IL-5, IL-10, and TNF-α), and/or by stimulating orpromoting wound healing.

The term “parenteral administration” as used herein includes any form ofadministration in which the compound is absorbed into the subjectwithout involving absorption via the intestines. Exemplary parenteraladministrations that are used in the present invention include, but arenot limited to intramuscular, intravenous, intraperitoneal,intraoccular, or intraarticular administration. Yet further, parenteraladministration also includes administration into a surgical field.

The term “pharmaceutically acceptable carrier” as used herein includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The term “pharmaceutical composition” as used herein refers to alactoferrin composition that this dispersed in a pharmaceuticallyacceptable carrier. The lactoferrin composition can comprise lactoferrinor an N-terminal lactoferrin variant in which at least the N-terminalglycine amino acid residue is truncated or substituted.

The term “oral administration” as used herein includes, but is notlimited to oral, buccal, enteral or intragastric administration.

The term “subject” as used herein, is taken to mean any mammaliansubject to which a human lactoferrin composition is orally administeredaccording to the methods described herein. In a specific embodiment, themethods of the present invention are employed to treat a human subject.Another embodiment includes treating a human subject suffering from awound.

The term “therapeutically effective amount” as used herein refers to anamount that results in an improvement or remediation of the symptoms ofthe disease or condition.

The term “topical administration” as used herein includes, but is notlimited to topical, dermal (e.g., trans-dermal or intra-dermal),epidermal, or subcutaneous.

The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of arecombinant human lactoferrin composition so that the subject has animprovement in the disease. The improvement is any improvement orremediation of the symptoms. The improvement is an observable ormeasurable improvement. Thus, one of skill in the art realizes that atreatment may improve the disease condition, but may not be a completecure for the disease.

The term “wound” as used herein refers to any injury, such as an ulcer,as a result of disease or disorder, or as a result of an accident,incident, or surgical procedure. Wound can be further defined as acuteand/or chronic.

B. Lactoferrin

The lactoferrin used according to the present invention can be obtainedthrough isolation and purification from natural sources, for example,but not limited to mammalian milk. The lactoferrin is preferablymammalian lactoferrin, such as bovine or human lactoferrin. In preferredembodiments, the lactoferrin is produced recombinantly using geneticengineering techniques well known and used in the art, such asrecombinant expression or direct production in genetically alteredanimals, plants or eukaryotes, or chemical synthesis. See, i.e., U.S.Pat. Nos. 5,571,896; 5,571,697 and 5,571,691, which are hereinincorporated by reference.

In certain aspects, the present invention provides lactoferrin variantshaving enhanced biological activities of natural LF and or rLF, e.g.,the ability to stimulate and/or inhibit cytokines or chemokines. Inparticular, the invention provides variants of lactoferrin from which atleast the N-terminal glycine residue has been substituted and/ortruncated. The N-terminal lactoferrin variants may occur naturally ormay be modified by the substitution or deletion of one or more aminoacids.

The deletional variants can be produced by proteolysis of lactoferrinand/or expression of a polynucleotide encoding a truncated lactoferrinas described in U.S. Pat. No. 6,333,311, which is incorporated herein byreference.

Substitutional variants or replacement variants typically contain theexchange of one amino acid for another at one or more sites within theprotein. Substitutions can be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

Still further, it is understood that an amino acid can be substitutedfor another having a similar hydrophilicity value and still obtains abiologically equivalent and immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

Thus, in the present invention, substitutional variants or replacementcan be produced using standard mutagenesis techniques, for example,site-directed mutagenesis as disclosed in U.S. Pat. Nos. 5,220,007;5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377; 5,789,166, and6,333,311, which are incorporated herein by reference. It is envisionedthat at least the N-terminal glycine amino acid residue can be replacedor substituted with any of the twenty natural occurring amino acids, forexample a positively charged amino acid (arginine, lysine, orhistidine), a neutral amino acid (alanine, asparagine, cysteine,glutamine, glycine, isoleucine, leucine, methionine, phenylaline,proline, serine, threonine, tryptophan, tyrosine, valine) and/or anegatively charged amino acid (aspartic acid or glutamic acid). Stillfurther, it is contemplated that any amino acid residue within the rangeof N1 to N16 can be replaced or substituted. It is envisioned that atleast up to 16 of the N-terminal amino acids residues can be replaced orsubstituted as long as the protein retains it biological and/orfunctional activity, which is stimulating the production of variouscytokines, such as IL-18, MIP-3α, GM-CSF or IFN-γ, or inhibitingproduction of various cytokines, such as IL-2, IL-4, IL-5, IL-10, andTNF-α, or promoting wound healing. Thus, the N-terminal lactoferrinvariants of the present invention are considered functional equivalentsof lactoferrin.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein is the concept that there is a limit to the numberof changes that may be made within a defined portion of the moleculewhile retaining a molecule with an acceptable level of equivalentbiological activity and/or enhancing the biological activity of thelactoferrin molecule. Biologically functional equivalents are thusdefined herein as those proteins in which selected amino acids (orcodons) may be substituted. Functional activity is defined as theability of lactoferrin to stimulate or inhibit various cytokines orchemokines and/or stimulate or promote wound healing.

Still further, the N-terminal amino acid residues can be substitutedwith a modified and/or unusual amino acids. A table of exemplary, butnot limiting, modified and/or unusual amino acids is provided hereinbelow.

TABLE A Modified and/or Unusual Amino Acids Abbr. Amino Acid Aad2-Aminoadipic acid BAad 3-Aminoadipic acid BAla beta-alanine,beta-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyricacid, piperidinic acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acidAib 2-Aminoisobutyric acid BAib 3-Aminoisobutyric acid Apm2-Aminopimelic acid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm2,2′-Diaminopimelic acid Dpr 2,3-Diaminopropionic acid EtGlyN-Ethylglycine EtAsn N-Ethylasparagine Hyl Hydroxylysine AHylallo-Hydroxylysine 3Hyp 3-Hydroxyproline 4Hyp 4-Hydroxyproline IdeIsodesmosine Aile allo-Isoleucine MeGly N-Methylglycine, sarcosine MeIleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

The presence and the relative proportion of an N-terminal lactoferrinvariants (deletions and/or subsititutions) in a preparation oflactoferrin (lactoferrin composition) may be done by determination ofthe N-terminal amino acid sequence by the process of Edman degradationusing standard methods. A relative proportion of N-terminal lactoferrinvariant comprises at least 1% of the lactoferrin composition, at least5% of the lactoferrin composition, at least 10% of the lactoferrincomposition, at least 25% of the lactoferrin composition, at least 50%of the lactoferrin composition or any range in between.

In this method, the protein is reacted with phenylisothiocyanate (PITC),which reacts with the amino acid residue at the amino terminus underbasic conditions to form a phenylthiocarbamyl derivative (PTC-protein).Trifluoroacetic acid then cleaves off the first amino acid as itsanilinothialinone derivative (ATZ-amino acid) and leaves the new aminoterminus for the next degradation cycle.

The percentage of N-terminal lactoferrin variant may also be done moreprecisely by using a Dansylation reaction. Briefly, protein isdansylated using Dansyl chloride reacted with the protein in alkalineconditions (pH 10). Following the Dansylation, the reaction mixtures aredried to pellets, then completely hydrolyzed in 6N HCl. The proportionof N-terminal amino acids are identified by RP HPLC using an in-linefluorometer in comparison with standards made up of known dansylatedamino acids.

C. Pharmaceutical Compositions

The present invention is drawn to a composition comprising a lactoferrincomposition that is dispersed in a pharmaceutical carrier. Thelactoferrin that is contained in the composition of the presentinvention comprises lactoferrin or an N-terminal lactoferrin variant inwhich at least the N-1 terminal glycine residue is truncated orsubstituted. More specifically, the N-terminal lactoferrin variantcomprises at least 1% of the composition, at least 5% of thecomposition, at least 10% of the composition, at least 25% of thecomposition, at least 50% of the composition or any range in between.

Yet further, the composition comprises lactoferrin in combination with ametal chelator dispersed in a pharmaceutical carrier. Thus, the presentinvention is drawn to a lactoferrin composition with or without a metalchelator that is dispersed in a pharmaceutical carrier. One of skill inthe art understands that both compositions (e.g., lactoferrin alone orlactoferrin in combination with a metal chelator) are within the scopeof the present invention and can be used interchangeably depending uponthe type of response that is desired. It is envisioned that the additionof a metal chelator to the lactoferrin composition enhances thesequestering of metal ions and thus strengthens the immune system orenhances the effect of lactoferrin.

Metal chelators that can be used in combination with lactoferrin,include the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or any salts thereof.More preferably, EDTA is used in combination with lactoferrin.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach or in the open wound environment. Examplesof stabilizers for use in an the composition include buffers, aminoacids such as glycine and lysine, carbohydrates such as dextrose,mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol,mannitol, etc., proteolytic enzyme inhibitors, and the like. Yetfurther, it is envisioned that divalent metal chelators, for exampleEDTA, can also be used to stabilize the composition of the presentinvention. More preferably, for an orally administered composition, thestabilizer can also include antagonists to the secretion of stomachacids. Yet further, for a topically administered composition, thestabilizer can also include antagonists to skin acids.

The composition for oral administration which is combined with asemi-solid or solid carrier can be further formulated into hard or softshell gelatin capsules, tablets, or pills. More preferably, gelatincapsules, tablets, or pills are enterically coated. Enteric coatingsprevent denaturation of the composition in the stomach or upper bowelwhere the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Uponreaching the small intestines, the basic pH therein dissolves thecoating and permits the lactoferrin composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells.

Further, the composition for topical administration which is combinedwith a semi-solid carrier can be further formulated into a gel ointment.A preferred carrier for the formation of a gel ointment is a gelpolymer. Gel polymers prevent denaturation of the composition in theopen skin by serum proteases. The gel formulation of the presentinvention also provides a controlled delivery system for lactoferrin orits activity on a wound site. Controlled delivery refers to drug releaseor activity release sufficient to maintain a therapeutic level over anextended period of time, such as up to 24 hours or more, preferably inthe range of 1 to 12 hours. The present gel formulation increases thecontact time of the lactoferrin at the wound site and provides asustained release dosage form necessary to achieve a significantincrease in the rate of wound healing. This is an important advantagebecause it permits less frequent application of the formulation to thewound and thereby permits fewer disturbances to the wound and itscellular components.

The gel formulation of the present invention has the advantage ofadhering to a wound and conforming to irregular body or wound contours.The gels may be applied directly to a wound site or in conjunction witha compliant porous or microporous substrate, for example in the form ofa coating, to be applied to the wound site. Gels have the furtheradvantages of having a high water content (which keeps the wound moist),the ability to absorb wound exudate, easy application to a wound andeasy removal by washing. Gels have a cool feeling when applied to awound and thus can increase patient comfort and acceptance of theformulation, especially on sensitive wounds.

The aqueous gels of the present invention have different viscositiesdepending on the intended application of the gel. Viscosity is a measureof the resistance of a liquid to flow. It is defined as the ratio of theshearing stress to the rate of shearing. The shear stress is theresistance of the liquid to flow under the influence of an appliedforce, i.e., the molecular resistance within a body opposing an externalforce. The shear stress is defined as the ratio of the force to the areasheared. When a liquid is sheared, assuming laminar flow, the layers ofthe liquid move at different rates. The relative rate of motion of thelayers is only one factor in the rate of shear. The other is thedistance, or clearance between the shearing planes. Thus, shear rate isdefined as the ratio of the velocity of the gel to the clearance.Viscosity has the dimensions of dynes/sec/cm². These dimensions arereferred to as poise. The dimensions of viscosity referred to herein,unless otherwise indicated, are in centipoise (cP) as measured using aBrookfield viscometer. All viscosity values are at room temperature,e.g., 22° C.-25° C., unless otherwise indicated.

The amount of lactoferrin in the present invention may vary from about 1μg to about 100 g of lactoferrin. In preferred embodiments, thecomposition of the present invention comprises a lactoferrinconcentration of about 0.0001% to about 30%. More preferably,lactoferrin is orally administered in the range of 10 mg to 25 g orlactoferrin is topically administered in the range of 1 μg to 5 g. Thelactoferrin may comprise lactoferrin or an N-terminal lactoferrinvariant in which at least the N-1 terminal glycine residue is truncatedand/or substituted.

More preferably, the composition of the present invention also containsmetal chelators, for example, but not limited toethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof. Theamount of the metal chelator in the composition may vary from about 1 ngto about 1 g. A preferred metal chelator is EDTA.

The gel forming materials of the present invention may be water-solublepolymers capable of forming a viscous aqueous solution or non-watersoluble, water swellable polymers (e.g., collagen), which can also forma viscous solution. Swellable polymers are those that absorb waterrather than dissolve in water. Cross-linked forms of the polymerdescribed herein may not be water soluble but may be water-swellable.Therefore, cross-linked forms of the polymer are within the scope of thepresent invention. Cross-linking refers to covalently bonding polymerchains together with a bifunctional reagent such as glutaraldehyde.Also, it is understood by those skilled in the art that certain polymersmay have to be used in the salt form or partially neutralized in orderto be made water soluble. For example, it is preferable to usehyaluronic acid as sodium hyaluronate to provide suitable watersolubility.

In the aqueous gel formulations for topical or incisional wound healing,the polymer may be selected from the group consisting of vinyl polymers,polyoxyethylene-polyoxypropylene copolymers, polysaccharides, proteins,poly(ethylene oxide), acrylamide polymers and derivatives or saltsthereof. It is understood that poly(ethyleneoxide) includes polyethyleneglycol. In the gel formulations for use in healing wounds in theanterior chamber of the eye, the polymers may be the same except that itis not preferred to use the polyoxyethylene-polyoxypropylene copolymersor poly(ethylene oxide). Also, for anterior chamber use, it is preferredthat the polymer is biodegradable, i.e., it will break down intoharmless constituents that can be drained from or metabolized in theanterior chamber. In the low viscosity, aqueous formulations for use inophthalmic wound healing, the gel forming polymers may be the same asfor topical or incisional wound healing, except that poly(ethyleneoxide) is not preferred to be used.

The vinyl polymers useful in the present invention may be selected fromthe group consisting of polyacrylic acid, polymethacrylic acid,polyvinyl pyrrolidone and polyvinyl alcohol. The polysaccharides usefulin the present invention are selected from the group consisting ofcellulose or cellulose derivatives, glycosaminoglycans, agar, pectin,alginic acid, dextran, starch, and chitosan. Starch occurs in two forms,α-amylose and amylopectin. The more water-soluble α-amylose ispreferred. The glycosaminoglycans are selected from the group consistingof hyaluronic acid, chondroitin, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparinsulfate and heparin. The glycosaminoglycans are used to enhance woundhealing in combination with any other gel-forming polymer. The proteinsuseful in the present invention are selected from the group consistingof collagen, gelatin and fibronectin. The acrylamide polymers arepolyacrylamide or polymethacrylamide polymers. Biocompatiblepolyacrylamide polymers are preferred. In further embodiments, carbomersare the preferred polyacrylamide polymer. Carbomers are synthetic highmolecular weight polymers of acrylic acid cross linked with either alkylesters of sucrose or pentaerythritol. Suitable commercially availablegrades of carbomer include Carbopol 910, Carbopol 934P, Carbopol 940,Carbopol 941, Carbopol 971P, Carbopol 974P, Carbopol 980, Carbopol 981,Carbopol 1342, Rheogic 252L, Rheogic 250H, and Hostacerin PN73.

In the gel formulation for topical or incisional wound healing, theviscosity may be within the range 1,000-12,000,000 cps at roomtemperature. It is preferred that the viscosity range be50,000-2,000,000. In one embodiment of the present invention, thetopical gel formulation may comprise 0.01-5% by weight polyacrylic acidhaving an average molecular weight of about 450,000-4,000,000. In apreferred embodiment, the polyacrylic acid is present at 0.5-1.5% byweight and has an average molecular weight of 2,000,000-4,000,000. ThepH of the polyacrylic acid gel should be within the range 4.5-8 and morepreferably in the range 6.5-7.5.

In another embodiment, the topical and incisional gel of the presentinvention may comprise 15-60% by weight of apolyoxyethylene-polyoxypropylene block copolymer having an averagemolecular weight of about 500-50,000. In a preferred embodiment, theblock copolymer is present at 15-40% by weight and has an averagemolecular weight in the range 1,000-15,000. The block copolymers used inthe present invention are commonly known as Pluronics. PreferredPluronics are Pluronic F88 and F127.

In a further embodiment, the topical or incisional gel may comprise 1 to20% by weight of a cellulose polymer having a molecular weight of about50,000 to 700,000. In a preferred embodiment, the cellulose polymer ispresent at 2-8% by weight and has an average molecular weight in therange 80,000-240,000. Preferred cellulose polymers arehydroxypropylmethyl cellulose (HPMC), carboxymethyl cellulose (CMC) andmethyl cellulose (MC).

In a further embodiment, the topical and incisional gel may comprise0.5-10% by weight of hyaluronic acid having an average molecular weightin the range 500,000 to 8,000,000. In a preferred embodiment, thehyaluronic acid is present at 1.5-6.0% by weight and the averagemolecular weight is greater than 1,000,000.

Acrylamide polymers may be useful for all types of wound healing,particularly in the anterior chamber of the eye. An absorbableacrylamide polymer, such as polyacrylamide, may be a good substitute forpresent carrier systems used in ophthalmic applications, such ashyaluronic acid. The acrylamide polymers may have an average molecularweight in the range 1-13 million, preferably about 4-6 million. Theweight percent of the acrylamide polymer in the gel may be 2-5%,preferably 3.5-4.5%. Substituted acrylamide polymers, such as methyl andalkyl substituted polymers are also within the scope of the presentinvention.

For use in the anterior chamber of the eye, an acrylamide gel deliverysystem has the following characteristics: any products of thedissolution or degradation of the delivery matrix are nontoxic and donot clog the trabecular mesh work; the gel is optically transparent; andthe gel can be left in the anterior chamber without causing adverseclinical effects such as an unacceptable increase in ocular pressure.

It will be readily apparent to one skilled in the art that the desiredviscosity range may be achieved by varying the molecular weight andpercent concentration of the polymer in the formulation. For example, agel having a low viscosity may be achieved by using a low molecularweight polymer or a lower percent concentration or a combination of thetwo. A high viscosity gel may be achieved by using a higher molecularweight polymer and a higher percent concentration. Intermediateviscosities may be achieved by varying the molecular weight and percentconcentration accordingly.

The low viscosity solution may comprise 0.01-2.0% by weight polyacrylicacid having an molecular weight of about 100,000-4,000,000. In apreferred embodiment, the polymer is present at 0.05-0.5%. In anotherembodiment, this dilute viscous solution may comprise 2-40% by weight ofa polyoxyethylene-polyoxypropylene copolymer having an average molecularweight of 500-500,000. Preferably, the concentration is 2-20% and themolecular weight is 1,000-15,000. Alternatively, the dilute viscoussolution may comprise a cellulose polymer at 1-20% and having amolecular weight of about 80,000-240,000. It is preferred that theconcentration be in the range of 1-10%. In a further embodiment, thedilute viscous solution may comprise 0.5-5.0% by weight hyaluronic acidhaving an average molecular weight of about 500,000-8,000,000.Preferably, the concentration is 0.5-2.0% and the average molecularweight is 1,000,000-6,000,000. If the dilute viscous solution is to beused as eye drops, it is preferred that the viscosity be in the range1-100 cps. If it is used for other applications, such as soaking abandage, then any viscosity in the range 1.0-5,000 will be suitable.

Upon formulation, solutions are administered in a manner compatible withthe dosage formulation and in such amount as is therapeuticallyeffective to result in an improvement or remediation of the symptoms.The formulations are easily administered in a variety of dosage formssuch as ingestible solutions, drug release capsules, gel ointments andthe like. Some variation in dosage can occur depending on the conditionof the subject being treated. The person responsible for administrationcan, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations meetsterility, general safety and purity standards as required by FDA Officeof Biologics standards.

D. Treatment of Wounds

In accordance with the present invention, a lactoferrin compositionprovided in any of the above-described pharmaceutical carriers isorally, topically, or parenterally administered to a subject suspectedof or having a wound. One of skill in the art can determine thetherapeutically effective amount of the composition to be administeredto a subject based upon several considerations, such as absorption,metabolism, method of delivery, age, weight, disease severity andresponse to the therapy. Oral administration of the composition includesoral, buccal, enteral or intragastric administration. It is alsoenvisioned that the composition may be used as a food additive. Forexample, the composition is sprinkled on food or added to a liquid priorto ingestion. Topical administration of the composition includestopical, dermal, epidermal, or subcutaneous administration. Parenteraladministration includes, but is not limited to intramuscular,intravenous, intraperitoneal, intraoccular or intraarticularadministration or administration into a surgical field.

The present invention is designed for the treatment of any type ofwound, which includes, but is not limited to skin wound, internal wound,gastrointestinal wound, oral wound, bone wounds, ophthalmic wound,surgical wound, or any combination thereof. Wounds can be found on butnot limited to skin, internal organs, stomach and intestines(gastrointestinal), oral mucosa, and eye (ophthalmic wounds, e.g.,corneal ulcers, radiokeratotomy, corneal transplants, epikeratophakiaand other surgically induced wounds in the eye). Depending on theprocess that causes the wounds, wounds can also be classified as but arenot limited to incisional wounds, excisional wounds, diabetic ulcers,venous stasis ulcers, decubitus or pressure ulcers, chemical wounds, andburn wounds.

A further embodiment of the present invention is administering theinventive composition to treat skin wounds. Skin wounds further comprisebut are not limited to full-thickness wounds and partial-thicknesswounds. Full-thickness wounds involve the complete removal of epidermisand dermis to the depth of fascial planes or subcutaneous fat. In theloose-skinned species, the thin musculature of the panniculus carnosus,which firmly adheres to the base of the dermis, is usually removed aswell. In partial-thickness wounds a substantial amount of dermis, mostlyreticular, is left behind, and, more importantly, the bases of mostepidermal appendages (sebaceous and sweat glands, hair follicles) remainintact.

Yet further, a wound can be further defined as an acute wound. Acutewounds have a relatively rapid rate of healing, especially in healthysubjects. However, in the elderly or immunocompromised healing can beprolonged. Healing is also prolonged if the wound becomes infected.Preferred acute wounds that are to be treated with the presentcomposition include, but are not limited to partial-thickness burns,lacerations, bullet wounds or infected wounds.

A wound is also further defined as a chronic wound. Examples of chronicwounds or chronic ulcers include, but are not limited to diabeticulcers, venous stasis ulcers, decubitus or pressure ulcers. Yet further,chronic wounds can also include infected wounds. Chronic wounds arewounds that do not repair or do so extremely slowly, and show partial ortotal lack of structural organization and functional coordination withnormal tissue. Chronic wounds or chronic ulcers can be broadlyclassified into three major types: diabetic ulcers, venous stasisulcers, decubitus or pressure ulcers. Diabetic ulcers often occur on afoot. Chronic diabetic state and poor glucose control results in poorperipheral circulation and microcirculation due to progressivearteriosclerosis; neuropathic changes that result in an insensateextremity prone to trauma; and intrinsic defects in the wound healingprocess that may include reduced abundance and response to cellulargrowth factors. In the case of venous ulcers, venous hypertension causesdisturbed microcirculation and pathological changes of the capillaries,elevated persistent levels of pro-inflammatory cytokines and proteases.Fibroblast senesce and respond less to growth factors, which distributeunfavorably. Proteolytic enzymes and their inhibitors are imbalanced.Pressure ulcers occur when skin is under pressure without movement toallow blood flow for 8-12 hours.

In a preferred embodiment of the present invention, the inventivecomposition (lactoferrin alone or lactoferrin in combination with ametal chelator) is administered in an effective amount to seal, toclose, to improve or to repair the wound. Also, it is envisioned thatthe composition of the present invention can also decrease, reduce, orinhibit, bacterial infections of the wound, which aid in the healingprocess of a wound.

Treatment regimens may vary as well, and often depend on wound type,wound location, wound and/or healing progression, and health and age ofthe patient. Obviously, certain types of wounds will require moreaggressive treatment, while at the same time, certain patients cannottolerate more taxing protocols. The clinician will be best suited tomake such decisions based on the known efficacy and toxicity (if any) ofthe therapeutic formulations.

In specific embodiments, the composition is given in a single dose ormultiple doses. The single dose may be administered daily, or multipletimes a day, or multiple times a week, or monthly or multiple times amonth. In a further embodiment, the composition is given in a series ofdoses. The series of doses may be administered daily, or multiple timesa day, weekly, or multiple times a week, or monthly, or multiple times amonth. Thus, one of skill in the art realizes that depending upon thewound type, location, health of the subject, etc., the lactoferrincomposition of the present invention may be administered for any givenperiod of time until the wound is healed at least by 5%, 10%, 20%, 30%,40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or anyrange in between.

For topical administration, the gel formulation of the present inventionmay be used to coat fibers of an absorbent gauze dressing to form awound healing bandage which may then be placed on a wound. The lowviscosity formulation is preferred for this use. The wound healingbandage may be prepared by soaking a gauze dressing with an aqueous gelsolution containing lactoferrin having wound healing activity. Thebandage can then be applied to the wound so that the coated fibers ofthe gauze contacts the wound and stimulate the rate of wound healing.

In those applications where the present invention is a gel that isapplied to an internal or incisional wound, it is preferred that the gelforming polymer be biodegradable. The naturally occurring polymers aregenerally biodegradable. Examples of these are collagen, theglycosaminoglycans, gelatin and starch. The cellulosics are notbiodegradable. The synthetic polymers such as the vinyl polymers are notdegradable. The biodegradability of the polymers described herein iswell known to those skilled in the art.

A further embodiment of the present invention is a method of treating awound comprising the step of supplementing the local immune system byincreasing the amount of lactoferrin in the vicinity of the wound.Preferably, the lactoferrin is administered topically to the wound.

Yet further, the present invention also provides a method of treating awound comprising the step of supplementing the systemic immune system byincreasing the amount of lactoferrin in the systemic circulation.Preferably, the lactoferrin is administered via a parenteral route,which includes, but is not limited to intramuscular, intravenous,intraperitoneal, intraoccular, intraarticular or into a surgical field.

Still yet, a further embodiment is a method of treating a woundcomprising the step of supplementing the mucosal immune system byincreasing the amount of lactoferrin in the gastrointestinal tract ofthe subject.

In further embodiments, the present invention provides a method ofenhancing the immune system of a subject suffering from a wound byadministering to the subject a lactoferrin composition. Depending uponthe mode of administration, different arms of the immune system areenhanced. For example, topical administration of the composition resultsin enhancement of the local immune system, i.e., in the vicinity of thewound. Parenteral administration of the composition results inenhancement of the systemic immune system. Yet further, oraladministration of the composition results in enhancement of the mucosalimmune system, which can also result in systemic effects as well. Infurther embodiments, the lactoferrin composition is administered incombination with a metal chelator, for example EDTA.

It is envisioned that the immune system, whether local, systemic ormucosal, is enhanced by lactoferrin stimulating cytokines and/orchemokines. Exemplary cytokines include interleukin-18 and GM-CSF in thegastrointestinal tract, which are known to enhance immune cells orstimulate production of immune cells. For example, interleukin-18enhances natural killer cells or T lymphocytes, which can kill bacteriainfecting a wound. In specific embodiments, interleukin-18 (IL-18)enhances CD4+, CD8+ and CD3+ cells. It is known by those of skill in theart that IL-18 is a Th₁ cytokine that acts in synergy withinterleukin-12 and interleukin-2 in the stimulation of lymphocyteIFN-gamma production. Other cytokines or chemokines may also be enhancedfor example, but not limited to IL-12, IL-1b, MIP-3α, MIP-1α, orIFN-gamma. Other cytokines or enzymes may be inhibited for example, butnot limited to IL-2, IL-4, IL-5, IL-10, TNF-α, or matrixmetalloproteinases. It is further contemplated that IL-18 or GM-CSFstimulate the production or activity of cells involved in wound repair,for example, but not limited to keratinocytes, endothelial cells,dendritic cells, fibroblasts, and myofibroblasts. Yet further, it isenvisioned that lactoferrin inhibits the production of TNF-alpha, whichinhibits cells involved in inflammation.

It is further envisioned that supplementing the local immune system in asubject by administering topically a therapeutically effective amount ofa lactoferrin composition in the vicinity of the wound can result in thekilling of bacteria infecting the wound. Still further, topicaladministration of a lactoferrin composition may stimulate the productionof a cytokine or a chemokine. Exemplary cytokines that can be stimulatedby lactoferrin include, but are not limited to interleukin-18 (IL-18),interleukin-12 (IL-12), granulocyte/macrophage colony-stimulating factor(GM-CSF), and gamma interferon (IFN-γ). Exemplary chemokines include,but are not limited to macrophage inflammatory protein 3 alpha (MIP-3α),macrophage inflammatory protein 1 alpha (MIP-1α), or macrophageinflammatory protein beta (MIP-1β).

The lactoferrin composition of the present invention can also result ininhibition of a cytokine or chemokine. The cytokines include, but arenot limited to interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5(IL-5), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α).Still further, the lactoferrin composition can also inhibit theproduction of matrix metalloproteinases (MMPs).

In further embodiments, cytokines, for example, interleukin-18 orgranulocyte/macrophage colony-stimulating factor, can stimulate theproduction or activity of immune cells. The immune cells include, butare not limited to T lymphocytes, natural killer cells, macrophages,dendritic cells, and polymorphonuclear cells. More specifically, thepolymorphonuclear cells are neutrophils and the T lymphocytes areselected from the group consisting of CD4+, CD8+ and CD3+ T cells.

In a further embodiment, cytokines, for example, interleukin-18 orgranulocyte/macrophage colony-stimulating factor, can also stimulate theproduction or activity of cells involved in wound repair. The cellsinvolved in wound repair include, but are not limited to keratinocytes,endothelial cells, fibroblasts, dendritic cells, and myofibroblasts. Theinhibition of TNF-alpha further inhibits the migration and maturation ofdendritic cells. The dendritic cells can be Langerhans cells.

E. Combination Treatments

In order to increase the effectiveness of the composition of the presentinvention, it may be desirable to combine the composition of the presentinvention with other agents effective in the treatment of wounds, suchas growth factors, skin replacement therapy, enzymatic and surgicaldebridement, moist wound dressings, cleansers, antibiotics. Such woundhealing agents are capable of negatively affecting a wound in a subject,for example, by enhancing the growth rate of skin cells, augmenting theblood supply to skin cells, promoting an immune response againstbacteria infecting the wound, killing bacteria, cleaning ischemictissue, promoting the closure of the wound. More generally, these otherwound healing agents are provided in a combined amount effective topromote the healing of a wound. This process may involve administeringthe composition of the present invention and the agent(s) or multiplefactor(s) at the same time. This may be achieved by administering asingle composition or pharmacological formulation that includes bothagents, or by administering two distinct compositions or formulations,at the same time, or at times close enough so as to result in an overlapof this effect, wherein one composition includes the lactoferrincomposition and the other includes the second agent(s).

Alternatively, the composition of the present invention may precede orfollow the other wound healing agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other wound healing agent andinventive composition are administered or applied separately to thewound, one would generally ensure that a significant period of time didnot expire between the time of each delivery, such that the agent andhuman lactoferrin composition would still be able to exert anadvantageously combined effect on the wound. In such instances, it iscontemplated that one may contact the wound with/administer bothmodalities within about 1-14 days of each other and, more preferably,within about 12-24 hours of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (2, 3,4, 5, 6, 7 or 8) lapse between the respective administrations.

1. Growth Factors

Wound healing therapies include growth factor based treatments. Examplesinclude, but are not limited to Regranex™ (Becaplermin-BB gel),AuTolo-Gel (autologous activated platelet releasate), Procuren(autologous thrombin-induced platelet releasate). Growth factors actwithout limitation by promoting granulation or the formation of newhighly vascularized connective tissue; stimulating proliferation,differentiation and migration of epithelial cells, vascular endothelialcells and other skin cells; enhancing the production of collagen,collagenase, and extracellular matrix.

2. Skin Replacement Therapy

Examples include but are not limited to Apligraf (bilayered livingskin), Trancyte (Human fibroblast-derived temporary skin substitute),Dermagraft (permanent, one-layer skin substitute), Epicel (livingone-layer artificial skin), Integra (collagen-based skin regenerationtemplate), AlloDerm (single-layer artificial skin made from humancadavers), CCS (living, cultured, artificial skin).

3. Enzymatic and Surgical Debridement

Debridement is a process or procedure to clean ischemic or dead tissue.Enzymatic debriders include Accuzyme papain-urea debriding ointment andCollagenase Santyl. Surgical debridement refers to physical removal ofat least part of the ischemic or dead tissue in a wound. Debridement maybe repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1,2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months. Enzymatic debridement treatments may be of varying dosages aswell. It is further contemplated that the present invention may be usedin conjunction with enzymatic or surgical debridement.

4. Dressings

Wound healing therapies include a variety of treatments based ondressings. Dressing categories include but are not limited to amorphoushydrogels, hydrogel sheets, absorptives, alginates, biological andsynthetic dressings, collagens, composites, contact layers, elasticgauzes, foams, gauzes and non-woven dressings, hydrocolloids,impregnated dressings, silicone gel sheets, silver dressings,transparent films, wound fillers

5. Cleansers

Examples include but are not limited to Biolex, Lamin, Wound WashSaline, Techni-Care, CarraKlenz, DiaB Klenz, MicroKlenz, RadiaCareKlenz, UltraKlenz, Comfee Sea-Clens, Optipore Sponge, Saf-Clens,Shur-Clens, Dermagran, DermaKlenz, Dumex, Gene Klenz, GRX, Allclenz,Restore, Hyperion, Medi Tech, Skintegrity, MPM Antimicrobial,ClinsWound, Septicare, Lobana Saline.

6. Antimicrobials

Examples of include but are not limited to Sulfamylon Cream, ThermazeneCream (1% silver sulfadiazine), cadexomer-iodine pads or gel. Examplesof intravenous antimicrobials include but are not limited toimipenem/cilastatin, β-lactam/ β-lactamase inhibitors(ampicillin/sulbactam, piperacillin/tazobactam), and broad-spectrumcephalosporins (cefoxitin, ceftizoxime, ceftazidime). Other examplesinclude, but are not limited to Bensal HP, Barri-Care, Care-Creme,Formula Magic, Baza, Micro-Guard, Ca-Rezz, Diabet-X products, MitrazolPowder, PiercingCare, Triple Care products, and various antifungalcreams and powders.

7. Compression

Dynamic compression examples, include pumps and sleeves such as but notlimited to ArtAssist, ArterialFlow, EdemaFlow, PulStar, Circulator Boot,Flowplus, Flowpress, Flowtron. Static compression include but are notlimited to leg wrappings, gloves, socks, leg wears, leg supports, armsleeves, stasis pads, compression hosieries, non-elastic bands, highcompression bandages, zinc impregnated bandages, elastic bandages.

8. Oxygen Therapy

Examples of systemic hyperbaric oxygen therapy include but are notlimited to compartments for one patient to lay down, for one patient tosit up to 25 degree angle, for one patient to sit up to 90 degree angle,for more than one patient to be treated simultaneously. Examples oftopical hyperbaric oxygen therapy include but are not limited todisposable topical hyperbaric oxygen systems for extremity ulcers,disposable topical hyperbaric oxygen systems for decubitis, post-op andtrauma wounds.

9. Hydrotherapy, Electric Therapy

Examples include but are not limited to dry hydrotherapy machines;non-contact thermal wound care systems for use on partial- andfull-thickness wounds that maintain warmth and humidity in the woundarea; systems that provide non-thermal, pulsed high frequency, high peakpower, electromagnetic energy to treat edema and pain in acute andchronic wounds; systems that use controlled, localized negative pressureand support for moist wound healing; pulsatile irrigators withcontrollable pressures below 15 psi for site-specific treatment ofvarious wounds with variety of tips; various wound irrigation andwhirlpool systems.

10. Nutritional Therapy Products

Examples include but are not limited to isotonic, high-protein,fiber-containing tube feedings to support wound healing; high-protein,cholesterol-free nutritional supplements.

11. Cohesives, Glues, Sealants, Patches

Examples include but are not limited to Dermabond, CoStasis, CoSeal,BioGlue, FibRx, FocalSeal, FloSeal, AutoSeal, Indermil, Syvek,LiquiSheild, LiquiBand, Quixil, CryoSeal, VIGuard Fibrin Sealant, andvarious tapes, closures, and securement products.

12. Topical Wound Healing Promoters

Examples include but are not limited to topical aerosols which stimulatethe capillary bed of chronic wounds; skin protectants with zinc-nutrientformulations; topical gels to help scars feel softer and smoother;hydrophilic ointments that cleanse degraded proteins, promote healthygranulation, control local inflammation and reduce wound odors;oil-and-water wound dressing emulsions that selectively recruitmacrophages.

13. Other Biotherapy Agents

Adjuvant therapy may also be used in conjunction with the presentinvention. The use of adjuvants or immunomodulatory agents include, butare not limited to tumor necrosis factor; interferon alpha, beta, andgamma; IL-2 and other cytokines; F42K and other cytokine analogs; orMIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines.

F. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Preparation of rhLF Carbomer Gels

Polyacrylic acid or Carbomer gels of grade 980 (Carbopol 980) wereprepared according to the present invention.

Six 30-gram rhLF gels were made having theoretical label strengths of0%, 0.1%, 0.3%, 1%, 2.5%, and 8.5%. The gels had the general formula asshown in Tables 1 and 2.

TABLE 1 General Formula of rhLF Gels 30 g batch % (ww) (g) PhosphateBuffer with rhLF** 86.6700 26.00** Carbopol 980 1.0000 0.30 EdetateDisodium, 0.1000 0.03 Dihydrate, USP Phenoxyethanol 1.0000 0.30Glycerin, USP 4.0000 1.20 Propylene Glycol, USP 5.0000 1.50 DimethicooneNF 350 0.4000 0.12 centistokes Citric Acid, Monohydrate 0.0956 0.03Granular, USP 20% Sodium Hydroxide q.s. to pH 6.5–7.5 q.s. to pH 6.5–7.6Purified Water, USP q.s. to 100% q.s. to 30 g **See Total Volume inTable 2

TABLE 2 Volume of Phosphate Buffer and rhLF Stock Used to Prepare theGels **Total rhLF wt Volume of Volume Volume in 30 g batch Stock ofPhos. Added RhLF (% w/w) (g) (ml)* Buf (ml) (ml) 0 0 0 26 26 0.1 0.030.3 25.7 26 0.3 0.09 039 25.1 26 1 0.3 3 23 26 2.5 0.75 7.5 18.5 26 8.52.55 25.5 0.5 26 **100 mg/mL rhLF in phosphate buffer Lot # E01764-03L

The gels were mixed in pre-weighed 125 ml stainless steel beakers andmixed using a programmable Caframo Mixer (Model BDC1850, Ontario,Canada). The usual mixing rate was 600 rpm using a stainless steelstirring rod with ½″ diameter blades. The gels were made by addingCarbopol 980 to phosphate buffer (6 mM sodium phosphate monobasicmonohydrate, 9 mM sodium phosphate dibasic heptahydrate, 50 mM NaCl, pH7). The volume of phosphate buffer used to make the initial gel is shownin the 4^(th) column (heading “Volume of Phos. Buf) in Table 2. AfterCarbopol 980 was fully hydrated (usually within 45 minutes), theglycerin, propylene glycol, and phenoxyethanol were added followed bycitric acid and then edetate disodium. Finally, the dimethicone wasadded. The pH of these gels was in the range of pH 3-3.5. At this point,20% sodium hydroxide was added to raise the pH. When the pH reachedabout 6, the rhLF was added (as shown in Table 2, 3^(rd) column “Volumeof Stock”). The pH was then raised to 7. The only deviation from theabove procedure was with the 8.5% w/w rhLF gel. Since the volume ofstock needed was large, the Carbopol 980 was added directly to 26 ml ofphosphate buffer containing rhLF. Thus, the rhLF in the 8.5% w/w/ gelwas exposed to a pH of 3-3.5 for a period of approximately 30-45minutes.

A Brookfield DV-III+ Rheometer was used to measure the viscosity of thegels in triplicate (0.5 ml of each gel). The conditions were:Temperature: 25° C.; Equilibration Time: 5 min; Spin Rate: 1.7 RPM; SpinTime: 5 min. Results are shown in Table 3.

TABLE 3 Gel Viscosity Average % rhLF Gel Rep 1 (cP) Rep 2 (cP) Rep 3(cP) Viscosity SD RSD Placebo 16634 16517 16751 16634 117 0.70 0.10%13190 13132 13482 13268 188 1.41 0.30% 16459 16400 16634 16498 122 0.741.00% 17509 17334 17626 17490 147 0.84 2.50% 19494 19611 19669 19591 890.46 8.50% 45641 45758 44240 45213 845 1.87

For the standard curve, 1.563-25 μg/ml rhLF standards were prepared bydiluting the 100 mg/ml rhLF stock (lot# E01764-03L) in de-ionized (DI)water. All active samples were taken from the top, middle, and bottom of3 ml aluminum crimped tubes and were prepared at a theoreticalconcentration of approximately 10 μg/ml rhLF in DI water. All activegels passed content uniformity specification (+/−10%).

EXAMPLE 2 Bioavailability of rhLF from Carbomer Gels on Full-Thickness,Open Wounds

Five Groups of three ICR male mice (weighing 22±2 gms) were used. Underhexobarbital (90 mg/Kg, IP) anesthesia, the shoulder and back region wasshaved. A sharp punch (ID 12 mm) was used to remove the skin includingpanniculus carnosus and adherent tissues.

A low and a high dose of recombinant human lactoferrin (rhLF)administered topically (carbopol-980 gel formulation) or one doseadministered intravenously (liquid solution) were applied immediatelyafter the injury, as indicated in Table 4. RhLF doses were given to20-gram mice immediately following wound injury. The following doseswere used: (1) Placebo, 0.04 ml/mouse, topical; (2) 50 mg/Kg, 0.04 ml ofa 2.5% gel; (3) 170 mg/Kg, 0.04 ml of an 8.5% gel. Three animals (onemale two females) were sacrificed at 0, 15, 30, 60, 120, and 240 minutesfollowing application of test compound. Sacrificed animals wereexsanguinated; blood was anticoagulated using EDTA, plasma was separatedfrom EDTA blood, and the samples were quickly frozen and stored at −80.

TABLE 4 Bioavailability of rhLF - Experimental Design and Methods PlasmaPreparation Minutes after Treatment Treatment Route Dose Sex 0 5 15 3060 120 240 None None 0 M A1 — — — — — — F A1 — — — — — — F A1 — — — — —— Vehicle TOP 0.04 M — B1 — — B2 B3 — (Placebo gel) ml/mouse F — B1 — —B2 B3 — F — B1 — — B2 B3 — PT# 1028377 TOP 50 M — C1 C2 C3 C4 C5 C6(AGX-6) mg/kg F — C1 C2 C3 C4 C5 C6 (rhLF gel) F — C1 C2 C3 C4 C5 C6 TOP170 M — D1 D2 D3 D4 D5 D6 mg/kg F — D1 D2 D3 D4 D5 D6 F — D1 D2 D3 D4 D5D6 PT#1 023296-ADD IV 5 M — E1 E2 E3 E4 E5 E6 (AGX-1) mg/kg F E1 E2 E3E4 E5 E6 (rhLF) F E1 E2 E3 E4 E5 E6

Concentrations of rhLF in plasma were determined using the BIOXYTECH®Lacto F EIA kit from OXIS Health Products, Inc, following the directionsprovided by manufacturer. Results were obtained by measuring absorbanceat 490 nm wavelength.

The peak plasma concentration attained was calculated relative to peakrhLF plasma concentrations following IV administration. The doseadjusted peak concentration of rhLF following topical gel application onopen full-thickness wounds at doses of 50 mg/Kg (151 ng/ml) and 170mg/Kg (75.1 ng/ml) was less than 0.5% of the normalized peakconcentration following 5 mg/kg IV rhLF (92,455.1 ng/ml) (see Table 5).The total plasma bioavailability, as calculated by the mean area underthe concentration curve (AUC) normalized to 170 mg/Kg, of the plasmarhLF time course with the 50 mg/Kg topical gel dose was 18.4 μg.min/ml,indicating an absolute systemic bioavailability of less than 0.5%. TheAUC for the 170 mg/Kg topical gel dose was 9.6 μg.min/ml, alsocorresponding to an absolute systemic bioavailability value of less than0.5% (See Table 5).

TABLE 5 Pharmacokinetics of topically-applied rhLF to open wounds inmice Low dose High dose topical topical Intravenous gel (50 gel (170rhLF solution mg/kg) mg/kg) (5 mg/kg) Peak plasma 44 75 2,719concentration (ng/mL) Time achieved (min) 60 60 5 Amount-normalized* 15175 92,456 (ng/mL) Normalized % of IV 0.2% 0.1% NA AUC (μg · min/mL) 5.49.6 152 AUC-N (μg · min/mL)** 18.4 9.6 5,174 Absolute bioavail- 0.4%0.2% NA ability *Values normalized to 170 mg/kg **AUC calculated withvalues normalized to 170 mg/kg.

EXAMPLE 3 Efficacy of rhLF Carbopol Gels in Wound Healing Experiments

RhLF carbopolymer gels were applied at concentration strengths of 2.5%and 8.5% directly to full thickness, open wounds in normal and diabeticdb/db mice. Diabetic (db/db) mice express lower levels of several growthfactors and receptors, accounting, at least in part, for a reduced rateof healing

Mice were anesthetized, the shoulder and back region of each animal wasshaved, and a sharp punch (ID 12 mm) was used to remove the skinincluding panniculus carnosus and adherent tissues (open, full-thicknesswounds). Different doses of rhLF were applied topically to the wounds(0.02 ml per wound) once per day for 11 days for normal mice or 20 daysfor diabetic db/db mice to compare the rates of healing with those ofnegative controls. The negative control was placebo gel. At several timepoints, the wound area was traced onto clear plastic and was measuredwith an Image Analyzer. The incidence of animals reaching 75% woundclosure was assessed and differences compared using Fisher's exact test.Differences were considered of statistical significance at p<0.05levels. Calculated time to 50% wound closure (CT-50) was measured viapolynomial equations (2 orders) and differences assessed with theStudent t test for significance.

FIG. 1 shows rhLF gels ranging from 0.1% to 8.5% mediated an improvementin the incidence of 75% wound closure of 77% in normal mice at day 12(p<0.01) and of 66% in diabetic db/db mice at day 15 (p<0.05). Table 6below shows individual values at each concentration for normal mice interms of incidence of 75% wound closure and calculated time to 50% woundclosure (CT-50).

TABLE 6 A Broad Range of RhLF Gel Strengths Accelerate Wound Healing inNormal Mic 75% Incidence on Day 12 Number of Percent % Healing Treatmentn Animals (%) on Day 12 CT50 (Days) Placebo gel 7 1 14 65.8 7.1 0.1% gel(0.02 mg) 7 7 100 (p = 0.0047) 90.6 (p < 0.0001) 4.4 (p = 0.0079) 0.3%gel (0.06) 7 6 86 (p = 0.0291) 86.5 (p = 0.001) 3.9 (p = 0.0507) 1.0%gel (0.2 mg) 7 5 71 (p = 0.1026) 88.6 (p = 0.001) 4.6 (p = 0.0392) 2.5%gel (0.5) 7 7 100 (p = 0.0047) 90.3 (p < 0.0001) 4.4 (p = 0.0148) 8.5%gel (1.7) 7 7 100 (p = 0.0047) 93.2 (p < 0.0001) 3.5 (p = 0.0024)

Based on these results, it is envisioned that topical, oral, orparenteral lactoferrin results in the killing of bacteria infecting awound, in the stimulation of IL-18, IL-12, GM-CSF, MIP-1α, MIP-1β,MIP-3α, or IFN-γ, and in the inhibition of IL-2, IL-4, IL-5, IL-10,TNF-α, or matrix metalloproteinases. It is further envisioned that IL-18or GM-CSF stimulate the production or activity of immune cells and cellsinvolved in wound repair, and that TNF-alpha inhibits cells involved ininflammation.

EXAMPLE 4 Wound Healing Time Course of rhLF, CGS-21680, and Regranex™ inNormal Mice

Groups of 7 ICR male mice were anesthetized, the shoulder and backregion of each animal was shaved, and a sharp punch (ID 12 mm) was usedto remove the skin including panniculus carnosus and adherent tissues.The wound area, traced onto clear plastic sheets on days 3, 5, 7, 9 and11 or 12, was quantitated with an Image Analyzer.

RhLF solution, vehicle (buffer), or a positive control (RhPDGF) wereapplied topically immediately following injury and once daily thereafterfor a total of 10 or 11 consecutive days. The unpaired Student's t testwas applied for comparison between treated and vehicle group at eachmeasurement time point. Differences were considered statisticalsignificant at P<0.05. RhPDGF (recombinant human Platelet Derived GrowthFactor-BB, Regranex™, becaplermin), which is presently the onlyavailable biological treatment in the market for chronic wounds(diabetic neuropathic ulcers), was used as a positive control at theapproved strength of 100 micrograms/gram (0.01%). CGS-21680 is an anadenosine A2A receptor agonist that was also used as a positive controlsince it was previously described as being very effective in promotingwound healing and in fact to promote more rapid wound healing thanRegranex™.

FIG. 2A indicates that rhLF exerted a comparable healing effect to thatof CGS-21680, suggesting that rhLF promotes more rapid wound repair thanRegranex™ (rhPDGF, Becaplermin). FIG. 2B confirms that rhLF promotes agreater extent of wound healing as compared to Regranex™.

Based on these results, it is envisioned that topical, oral, orparenteral lactoferrin results in the killing of bacteria infecting awound, in the stimulation of IL-18, IL-12, GM-CSF, MIP-1α, MIP-1β,MIP-3α or IFN-γ, and in the inhibition of IL-2, IL-4, IL-5, IL-10,TNF-α, or matrix metalloproteinases. It is further envisioned that IL-18or GM-CSF stimulate the production or activity of immune cells and cellsinvolved in wound repair, and that TNF-alpha inhibits cells involved ininflammation.

EXAMPLE 5 Efficacy of rhLF Topical Solution vs. Regranex™ in WoundHealing Experiments

Mice were anesthetized, the shoulder and back region of each animal wasshaved, and a sharp punch (ID 12 mm) was used to remove the skinincluding panniculus carnosus and adherent tissues (open, full-thicknesswounds). Different doses of rhLF were applied topically to the wounds(0.02 ml per wound) once per day for 11 days for normal mice or 20 daysfor diabetic db/db mice to compare the rates of healing with those ofnegative and positive controls. Negative control or placebo was rhLFvehicle (a PBS solution). Positive control was RhPDGF (recombinant humanPlatelet Derived Growth Factor-BB, Regranex™, becaplermin) used at theapproved strength of 100 micrograms/gram (0.01%).

RhPDGF (recombinant human Platelet Derived Growth Factor-BB, Regranex™,becaplermin), an approved drug for treatment of chronic diabetic ulcers,was used as a positive control at the approved strength of 100micrograms/gram (0.01%). At several time points, the wound area wastraced onto clear plastic and was measured with an Image Analyzer. Theincidence of animals reaching 75% wound closure was assessed anddifferences compared using Fisher's exact test. Differences wereconsidered of statistical significance at p<0.05 levels.

FIG. 3 shows pooled data from the 5 experiments, demonstrating thesuperior efficacy of rhLF compared to Regranex™ for wound repair atdoses ranging from 0.1% to 10%. Animals treated with rhLF 0.1%-10% (147animals) had a 34% increase in the incidence of 75% wound closure on thefinal day of the experiment relative to placebo (42 animals, p<0.0001)and a 32% increase relative to Regranex™ (21 animals, p<0.001). Inhealthy mice, 1% rhLF significantly (p<0.01) increased this parameterrelative to placebo 38% and relative to becaplermin (Regranex™) 36%(p<0.01). In diabetic db/db mice with impaired wound repair function, 1%rhLF gel increased the incidence of 75% wound closure on day 15 by 83%over placebo (p<0.01).

Based on these results, it is envisioned that topical, oral, orparenteral lactoferrin results in the killing of bacteria infecting awound, in the stimulation of IL-18, IL-12, GM-CSF, MIP-1α, MIP-1β,MIP-3α or IFN-γ, and in the inhibition of IL-2, IL-4, IL-5, IL-10,TNF-α, or matrix metalloproteinases. It is further envisioned that IL-18or GM-CSF stimulate the production or activity of immune cells and cellsinvolved in wound repair, and that TNF-alpha inhibits cells involved ininflammation.

EXAMPLE 6 Efficacy of Oral rhLF in Wound Healing Experiments

Mice were anesthetized, the shoulder and back region of each animal wasshaved, and a sharp punch (ID 12 mm) was used to remove the skinincluding panniculus carnosus and adherent tissues. Different doses ofrhLF were applied topically to the wounds once per day for 11 days fornormal mice or 20 days for diabetic db/db mice to compare the rates ofhealing with those of negative controls. Negative control or placebo wasrhLF vehicle (a PBS solution). At several time points, the wound areawas traced onto clear plastic and was measured with an Image Analyzer.The incidence of animals reaching 75% or 100% wound closure was assessedand differences compared using Fisher's exact test. Differences wereconsidered of statistical significance at p<0.05 levels.

At several time points, the wound area was traced onto clear plastic andwas measured with an Image Analyzer. The incidence of animals reaching75% wound closure was assessed on days 9-12 for normal mice and days 15or 19 for diabetic mice, and differences compared using Fisher's exacttest. Differences were considered of statistical significance at p<0.05levels.

FIG. 4A shows that oral rhLF given to healthy mice at doses ranging from0.5 to 4.5 mg/Kg resulted in an improvement of 43% in the incidence of75% wound closure compared to oral placebo, with the highest doseshowing a 52% improvement (p<0.01). FIG. 4B shows that oral rhLF givento diabetic db/db mice at 4.5 to 65 mg/Kg doses of rhLF increased theincidence of 75% wound closure on day 15 by 75% over placebo with thehighest dose tested achieving an increase of 83% over placebo (p<0.01).Similarly, FIG. 4C indicates that 4.5 to 65 mg/Kg doses of oral rhLF todiabetic db/db mice achieved 75% increase in incidence of 100% woundclosure by day 19 with the highest dose reaching an increase of 100%compared to placebo (p<0.01).

Based on these results, it is envisioned that topical, oral, orparenteral lactoferrin results in the killing of bacteria infecting awound, in the stimulation of IL-18, IL-12, GM-CSF, MIP-1α, MIP-1β,MIP-3α or IFN-γ, and in the inhibition of IL-2, IL-4, IL-5, IL-10,TNF-α, or matrix metalloproteinases. It is further envisioned that IL-18or GM-CSF stimulate the production or activity of immune cells and cellsinvolved in wound repair, and that TNF-alpha inhibits cells involved ininflammation.

EXAMPLE 7 Topical and Oral rhLF Wound Healing Experiment in InfectedWounds

The efficacy of topical rhLF in bacteria-infected wounds was tested.This animal model represents a clinically relevant situation sincediabetics often have infected ulcers and such infection is believed tocontribute to the impairment of wound repair. Staphylococcus aureus isone of the most common bacteria infecting the diabetic foot ulcer and isassociated with an increase in mortality rate.

Groups of 7 ICR male mice were anesthetized, the shoulder and backregion of each animal was shaved, and a sharp punch (ID 12 mm) was usedto remove the skin including panniculus carnosus and adherent tissues.Immediately after puncture, a 9.6×10⁵ CFU/0.02 ml/mouse ofStaphylococcus aureus (Smith) was applied to the wound region of eachanimal. The wound area, traced onto clear plastic sheets on days 3, 5,7, 9 and 12, was quantitated with an Image Analyzer.

Topical rhLF, oral rhLF, vehicle (buffer), or a positive control(Regranex™ 0.01%) were applied immediately following injury and bacteriaand once daily thereafter for a total of 11 consecutive days. Forlactoferrin applied orally, mice were given 0.130 ml of the rhLFsolution via gavage (directly feeding the animal with a flexible tube).The incidence of animals reaching 75% wound closure was assessed anddifferences compared using Fisher's exact test. Differences wereconsidered of statistical significance at P<0.05 levels.

FIG. 5A and FIG. 5B shows that topical rhLF increased the incidence of75% closure by 86% relative to placebo (p<0.01) and 71% relative toRegranex™ (p<0.05). FIG. 5B shows that oral rhLF improved the incidenceof 75% closure by 72% relative to placebo p<0.05).

Based on these results, it is envisioned that topical, oral, orparenteral lactoferrin results in the killing of bacteria infecting awound, in the stimulation of IL-18, IL-12, GM-CSF, MIP-1α, MIP-1β,MIP-3α or IFN-γ, and in the inhibition of IL-2, IL-4, IL-5, IL-10, TNF-α, or matrix metalloproteinases. It is further envisioned that IL-18 orGM-CSF stimulate the production or activity of immune cells and cellsinvolved in wound repair, and that TNF-alpha inhibits cells involved ininflammation.

EXAMPLE 8 Best Topical rhLF Dose Determination

The experimental protocol in Example 5 is used to determine the lowestoral rhLF dose that promotes wound healing in mice. Smaller doses ofrhLF are systematically tested until no further wound repair effect isapparent. EDTA is added in an attempt to increase the potency of rhLFand further decrease the rhLF dose that is effective. Topical vehicle isused as the negative control.

EXAMPLE 9 Best Oral rhLF Dose Determination

The experimental protocol in Example 5 is used to determine the lowestoral rhLF dose that promotes wound healing in mice. Smaller doses ofrhLF are systematically tested until no further wound repair effect isapparent. EDTA is added in an attempt to increase the potency of rhLFand further decrease the rhLF dose that is effective. Oral vehicle isused as the negative control.

EXAMPLE 10 Combination Study

The experimental protocol in Example 5 is used to determine the rate ofwound healing in mice. Combinations of oral with topical rhLF and oralrhLF with topical Regranex™ are tested with and without EDTA. Oralvehicle plus placebo gel, and oral vehicle plus Regranex™ are thenegative and positive controls, respectively. Oral rhLF plus placebogel, and oral vehicle plus topical rhLF are the synergy controls.

EXAMPLE 11 Efficacy Comparison of RhLF Gel and Liquid SalineFormulations in Wounds Covered with Dressings

The experimental protocol in Example 5 is used to determine the rate ofwound healing in mice. Liquid formulations of rhLF, 0.2 mg/ml (0.02 mlof a 10 mg/ml solution) are applied to the wound. The wound area is thencovered with a saline-moistened gauze dressing. This process is repeateddaily for 10 days. RhLF Gel 0.2 mg [0.020 mL of a 10 mg/ml gel] isapplied directly to the wounds and covered with a dressing, daily, for10 days. 0.02 ml of rhLF solution vehicle and placebo gel are applied towounds and covered with dressing, daily, for 10 days. Regranex™ is thepositive control and is applied topically, 0.02 ml (100 μg/ml clinicalconcentration), in a similar fashion.

EXAMPLE 12 Efficacy of Different Application Regimens of rhLF Gel andDressings

The experimental protocol in Example 5 is used to determine the rate ofwound healing in mice. RhLF Gel 0.2 mg [0.020 mL of a 10 mg/ml gel] isapplied directly to the wounds and covered with a saline-moistened gauzedressing, daily, for 10 days. In another group of mice the dressing ischanged every other day. In a third group, Regranex™: rhLF gel isapplied to the wound and covered with a dressing. After 12 hours, theulcer is rinsed gently with saline or water to remove residual gel, andthe wound is covered with a fresh dressing for an additional 12 hours.Placebo gel 0.02 ml and Regranex™ 0.02 ml (100 μg/ml), are the negativeand positive controls, respectively, and are applied using the latterregime.

EXAMPLE 13 RhLF Gel: Comparison of Once Daily to Twice Daily

The experimental protocol in Example 5 is used to determine the rate ofwound healing in mice. RhLF Gel 0.2 mg/wound/day [0.020 mL of a 10 mg/mlgel] is applied directly to the wounds, daily, for 10 days. To a secondgroup of animals, 0.1 mg twice daily (BID) is applied, each applicationseparated by a 12 hour interval. Placebo gel 0.02 ml and Regranex™, 0.02ml (100 ug/ml), applied once per day, are the negative and positivecontrols, respectively.

EXAMPLE 14 Efficacy of Topical rhLF Alone or in Combination withRegranex™

The experimental protocol in Example 5 is used to determine the rate ofwound healing in mice. RhLF Gel 0.2 mg/wound/day [0.020 mL of a 10 mg/mlgel] is applied directly to the wounds daily, for 10 days. A secondgroup of mice receives rhLF 0.2 mg/wound and Regranex™ 2 ug/wound (0.02ml/wound, 10 ug/ml). A third group gets rhLF 0.02 mg/wound and Regranex™0.002 ug/wound to test for potential synergies. Placebo gel 0.02 ml,placebo plus Regranex™ 0.02 ml (100 ug/ml), and Regranex™ alone, are thenegative, synergy, and positive controls, respectively.

EXAMPLE 15 Dose Escalation, Pharmacokinetic and Pharmacodynamic Trial ofTopical rhLF in Patients with Diabetic Ulcers

This is a Phase I/II 14-day dose escalation study in humans designed toevaluate the escalating dosing regimens of topical rhLF gel in patientswith chronic diabetic ulcers and determine a maximally tolerated dose(if any) for 1% up to 8.5% rhLF concentration. RhLF is evaluated for itsability to promote ulcer healing.

EXAMPLE 16 Dose Escalation, Pharmacokinetic and Pharmacodynamic Trial ofOral rhLF in Patients with Diabetic Ulcers

Study design is similar to the one in Example 14, except that the rhLFis applied orally with and without EDTA.

EXAMPLE 17 Trial of rhLF versus placebo and Standard-of-Care in Patientswith Diabetic Ulcers

This is a randomized, double-blind, placebo-controlled, multicenterstudy in humans. It is a 12-week phase II clinical trial in patientswith diabetic chronic ulcers to evaluate the efficacy of treatment withtwo dose levels of a topical or oral administration of rhLF incomparison with placebo, Regranex™, and standard-of care only. Efficacyis evaluated by incidence of partial and complete wound closure, andtime to healing.

EXAMPLE 18 Release of rhLF from the Carbomer Gels

RhLF-Carbopol-980 gels at several concentration strengths are tested forrelease kinetics of rhLF over time from the dosage form and are detectedin a receiving buffer in an in vitro diffusion system.

EXAMPLE 19 Additional Studies of rhLF-Vinyl Polymer Gels

RhLF containing gels are prepared based on polymethacrylic acid,polyvinyl pyrrolidone and polyvinyl alcohol polymers. Viscosity of gelsis determined via a Brookfield DV-III+Rheometer and protein contentuniformity is measured using the BCA assay, as described in example 1.Bioavailability is assessed following application of these vinyl polymergels on open, full-thickness wounds in normal mice, as described inexample 2. Efficacy of wound healing activity of these vinyl polymer gelformulations is tested in mouse model as described in examples 3-5.Several concentration strengths of these vinyl polymer gels are testedfor release kinetics of rhLF over time from the dosage form and aredetected in a receiving buffer in an in vitro diffusion system.

EXAMPLE 20 Studies of rhLF-Polysaccharide Polymer Gels

RhLF-Polysaccharide polymer gel formulations are prepared. Viscosity ofgels is determined via a Brookfield DV-III+Rheometer and protein contentuniformity is measured using the BCA assay, as described in example 1.Bioavailability is assessed following application of the polysaccharidepolymer gels on open, full-thickness wounds in normal mice, as describedin example 2. Efficacy of wound healing activity of the polysaccharidepolymer gel formulations is tested in mouse model as described inexamples 3-5. Several concentration strengths of the polysaccharidepolymer gels are tested for release kinetics of rhLF over time from thedosage form and are detected in a receiving buffer in an in vitrodiffusion system.

EXAMPLE 21 Studies of rhLF-Glycosaminoglycan Polymer Gels

RhLF-Glycosaminoglycan polymer gel formulations are prepared. Viscosityof gels is determined via a Brookfield DV-HIII+Rheometer and proteincontent uniformity is measured using the BCA assay, as described inexample 1. Bioavailability is assessed following application of theseglycosaminoglycan polymer gels on open, full-thickness wounds in normalmice, as described in example 2. Efficacy of wound healing activity ofthe glycosaminoglycan polymer gel formulations is tested in mouse modelas described in examples 3-5. Several concentration strengths of theglycosaminoglycan polymer gels are tested for release kinetics of rhLFover time from the dosage form and are detected in a receiving buffer inan in vitro diffusion system.

EXAMPLE 22 Studies of rhLF-Protein Polymer Gels

RhLF-Protein polymer gel formulations are prepared. Viscosity of gels isdetermined via a Brookfield DV-III+Rheometer and protein contentuniformity is measured using the BCA assay, as described in example 1.Bioavailability is assessed following application of these proteinpolymer gels on open, full-thickness wounds in normal mice, as describedin example 2. Efficacy of wound healing activity of the protein polymergel formulations is tested in mouse model as described in examples 3-5.Several concentration strengths of the protein polymer gels are testedfor release kinetics of rhLF over time from the dosage form and aredetected in a receiving buffer in an in vitro diffusion system.

EXAMPLE 23 Studies of rhLF-Pluronic Polymer Gels

RhLF-Pluronic polymer gel formulations are prepared. Viscosity of gelsis determined via a Brookfield DV-III+Rheometer and protein contentuniformity is measured using the BCA assay, as described in example 1.Bioavailability is assessed following application of these pluronicpolymer gels on open, full-thickness wounds in normal mice, as describedin example 2. Efficacy of wound healing activity of the pluronic polymergel formulations is tested in mouse model as described in examples 3-5.Several concentration strengths of the pluronic polymer gels are testedfor release kinetics of rhLF over time from the dosage form and aredetected in a receiving buffer in an in vitro diffusion system.

EXAMPLE 24 Studies of rhLF-Acrylamide Polymer Gels

RhLF-Acrylamide polymer gel formulations are prepared. Viscosity of gelsis determined via a Brookfield DV-III+Rheometer and protein contentuniformity is measured using the BCA assay, as described in example 1.Bioavailability is assessed following application of these acrylamidepolymer gels on open, full-thickness wounds in normal mice, as describedin example 2. Efficacy of wound healing activity of the acrylamidepolymer gel formulations is tested in mouse model as described inexamples 3-5. Several concentration strengths of the acrylamide polymergels are tested for release kinetics of rhLF over time from the dosageform and are detected in a receiving buffer in an in vitro diffusionsystem.

EXAMPLE 25 Wound Healing Rates With RhLF Preparations Differing inProportion of N-1 Truncate

The biological activity of preparations of rhLF differing in thepercentage of N-1 truncates was compared using a mouse model of woundhealing. Groups of seven mice were anesthetized, the shoulder and backregion of each animal was shaved, and a sharp punch (ID 12 mm) was usedto remove the skin including panniculus carnosus and adherent tissues(open, full-thickness wounds). Either rhLF (20 microgram per mouse) orplacebo was applied topically to the wounds once per day for 11 days. Atseveral time points, the wound area was traced onto clear plastic andwas measured with an Image Analyzer. The CT50 (time to 50% closure) wasassessed. (Experiments AN-W2, W3, W9).

As shown in Table 7, rhLF preparations containing entirely intactprotein as well as those containing N-1 truncates ranging from 27% to42% all showed efficacy relative to their respective placebos that wasstatistically significant. Percent improvement between the batches wascomparable and statistically indistinguishable.

TABLE 7 Wound Healing Rates With Three Different RhLF Preparations RhLFCT50 Percent Percent N-1 Preparation (Active/Placebo) ImprovementTruncate 7001 4.8/5.9 Days 19%  0% L005 6.0/7.6 Days 21% 27% L0075.3/6.4 Days 17% 42%

EXAMPLE 26 Wound Closure Incidence With RhLF Preparations Differing inProportion of N-1 Truncate

In the experiments described in Example 25, incidence of 80% woundclosure was measured in mice treated with placebo or rhLF. The absoluteincrease in the incidence of 80% wound closure in rhLF treated animalsrelative to placebo animals in the same experiment was determined on Day9 (7001 and L007) or Day 12 (L005).

As shown in Table 8, rhLF preparations containing entirely intactprotein as well as those containing N-1 truncates ranging from 27% to42% all showed efficacy with an increased wound closure that wasstatistically significant with respect to their respective placebos.

TABLE 8 Wound Closure Incidence With Three Different RhLF PreparationsRhLF 80% Closure Percent Percent N-1 Preparation (Active/Placebo)Improvement Truncate 7001 86%/57% 29%  0% L005 43%/0%  43% 27% L00743%/0%  43% 42%

REFERENCES CITED

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

1. A method of treating a diabetic ulcer, comprising the step ofadministering to a subject having a diabetic ulcer a therapeuticallyeffective amount of a lactoferrin composition to result in animprovement or a remediation of a symptom of the diabetic ulcer.
 2. Themethod of claim 1, wherein said lactoferrin composition is administeredtopically, orally or parenterally.
 3. The method of claim 2, whereinsaid lactoferrin composition is administered orally.
 4. The method ofclaim 3 further comprising administering an antacid in conjunction withsaid lactoferrin composition.
 5. The method of claim 2, wherein saidlactoferrin composition is administered topically.
 6. The method ofclaim 2, wherein said lactoferrin composition is administeredparenterally.
 7. The method of claim 1 further comprising administeringa standard wound healing therapy in combination with the lactoferrincomposition.
 8. The method of claim 1, wherein the administeringcomprises administering said composition for at least one week to atleast twelve weeks.
 9. The method of claim 1, wherein the amount of thelactoferrin that is administered is about 0.0001 μg to about 100 g perday.
 10. The method of claim 1, wherein said composition is a topicalgel, a solution, capsule or a tablet having a lactoferrin concentrationof about 0.0001% to about 30%.
 11. The method of claim 10, wherein saidtopical gel is composed from a polymer selected from the group ofconsisting of a vinyl polymer, polysaccharide polymer, glycosaminoglycanpolymer, protein polymer, polyoxyethylene-polyoxypropylene polymer,carbomers and acrylamide polymer.
 12. The method of claim 11, whereinthe polymer concentration is about 0.5% (w/w) to about 3.0% (w/w) andthe polymer has a molecular weight of about 50,000 to about 13,000,000.